Conceiving and rearing children is one of the most pleasurable and rewarding aspects of life for many people. Unfortunately, numerous couples find themselves infertile with one another, unable to conceive without technological assistance. Such couples can be aided by a variety of technological methods such as artificial insemination (intracervical, intrauterine (IUI), intratubular and direct intraperitoneal (DIPI) insemination), gamete intrafallopian transfer (GIFT), in vitro fertilization and embryo transfer (IVFET), zygote intrafallopian transfer (such as ZIFT, PROST and TET), peritoneal oocyte and sperm transfer (POST), and sex selection, among others. However, these methods are generally costly, due to the high degree of training required of the medical participants, and the significant possibility of failure upon a single attempt at fertilization.
One source of the costs of these methods is the need to isolate higher viability spermatozoa, that is, spermatozoa having relatively higher motility and relatively more normal membranes, during the practice of these methods. Higher viability is believed to correlate to higher fertilizing capacity. Moreover, typical semen samples will contain materials which are known to interfere with the successful fertilization of an ovum, and with the successful maintenance of the fertilized ovum in the female patient. Such materials include paternal plasma, protein, leukocytes, spermdecapitation factors and other extraneous materials, and dead, agglutinated or nonviable spermatozoa (those spermatozoa having relatively low motility or possessing unhealthy, damaged or abnormal membranes, in particular,membranes which do not swell in hypotonic solutions). If not filtered from the spermatozoa-containing fluid used for fertilization, these materials can induce an adverse reaction in the female patient and result in spontaneous abortion of the fertilized ovum. For example, seminal plasma can cause severe uterine cramping (due to prostaglandins) when placed in the uterus during IUI. Similar problems are encountered with the artificial insemination of other mammals, e.g., bovines or equines, as well as other animals (e.g., turkeys). In short, higher viability spermatozoa appear more likely to yield successful fertilization and impregnation, and more likely to survive cryopreservation, and are therefore desirable in fertility techniques.
Numerous methods for separating higher viability spermatozoa from lower viability spermatozoa or other undesirable materials are known, and each has its advantages and drawbacks. For example, while the well-known swim up, swim down and Percoll density gradient centrifugation techniques obtain highly motile spermatozoa populations, the yields of these techniques is generally poor. This is particularly disadvantageous when the original ejaculates are oligozoospermic or asthenozoospermic.
Another example is given in U.S. Pat. No. 4,999,283 (Zavos et al.), which incidentally discloses the filtering of a spermatozoa-containing fluid through a "Sephadex" column as an adjunct to a filtering technique for separating male and female determining spermatozoa (column 6,lines 35 through 45). ("Sephadex" is believed to be a trademark for synthetic microscopic beads composed of crosslinked dextran.) The "Sephadex" is retained in the column (three disposable plastic syringes) by pads of glass wool, specifically, Johns-Manville (Denver, Colo.) MicroFiber, code 112, No. 475 glass. As disclosed at column 7, line 44 through column 8, line 18, the pads are prepared by taking thin sheets of 3 to 4 mg fiber and folding the long fibers under to make a circle about 1 cm in diameter, and the pads are then gently pushed into the bottom of the syringes (using a Pasteur pipet) until 1 to 2 mm protrudes through the holes in the syringes. The density of the glass wool, as indicated by flow rates, is adequate to prevent "Sephadex" from passing into the collection vials, yet not so much as to result in insufficient filtering. (More detail on the procedure for packing and using "Sephadex" columns can be found at page 71 of Graham et al., "An Overview of Column Separation of Spermatozoa," in Proc. 7th Tech. Conf. A.I. Reprod., NAAB, Madison, Wis., Apr. 14-15, 1978.) Clearly, the method contemplates that the successful discrimination between viable and nonviable sperm is achieved by the "Sephadex" beads, and not by the glass wool itself.
Glass wool has been found by itself, however, to be quite useful for separating higher viability spermatozoa from lower viability spermatozoa and other undesired materials. The present inventor has found good success with a modification of a method originally reported by Paulson and Polakoski (ibid, page 70, citing Fertil. Steril. 28:178-181 (1977)). As described in a continuing series of articles, beginning with Jeyendran et al., "Concentration of Viable Spermatozoa for Artificial Insemination," Fertil. Steril. 45(1):132-134 (1986), when the technique of glass wool filtration is standardized, it yields a higher recovery of viable spermatozoa than either the swim up or Percoll density gradient techniques, is significantly less time consuming and less costly in personnel training and equipment than either, and yields spermatozoa capable of penetrating a significantly higher percentage of denuded hamster oocytes (determined by the human sperm denuded hamster oocyte penetration (SPA) system) and of fertilizing a significantly higher percentage of intact human oocytes than either technique. The technique as standardized by Jeyendran employs 30 mg of glass wool microfiber manually positioned in the bottom of a 3 ml syringe barrel, carefully packed to a depth of 3 mm.
The Zavos et al. and Jeyendran techniques are subject to some drawbacks, however. In each, the density of the packed glass wool is critical to the success of the technique. Unfortunately, it takes significant time to train medical personnel to properly pack the glass wool in the filter columns, it takes significant time to perform the packing for numerous samples, and the personnel performing the packing must have a reasonable degree of familiarity with laboratory technique. These all add to the cost of performing the separation.
The problem of nonuniform density of the glass wool leads to other problems with each technique. In the technique of Zavos et al., the glass wool can either pass the "Sephadex" or fail to pass the sample, which in either case can require preparation of another sample, and at a minimum, a repeat of the filter preparation and filtration itself. In the technique of Jeyendran, even when preparation of the glass wool filter is standardized, the filter still allows a significant percentage (typically, on the order of 25 percent) of the lower viability spermatozoa to pass through. This may arise from two causes. First, it is likely that some lower viability spermatozoa are able to pass around the glass wool, that is between the glass wool and the inside of the filter assembly. Second, due to the resilient nature of the glass wool fibers, the volume of wool can change after the filter assembly is packed, thus decreasing the effective density of the glass wool and decreasing the efficacy of the filtration technique.
It should therefore be clear that it would be highly desirable to discover a technique or device which enjoys the successes of the Jeyendran technique and yet is easy and inexpensive to manufacture; and which simultaneously achieves the desired density of glass wool as employed in the Jeyendran technique and can handle fluid samples which are low in volume (important for enabling deep intracervical or intrauterine insemination), yet avoids the problems encountered with the variety of techniques described above.